jeremy max levin, erich jost and peter richard cook- the dissociation of nuclear proteins from...

8/3/2019 Jeremy Max Levin, Erich Jost and Peter Richard Cook- The Dissociation of Nuclear Proteins from Superhelical DNA

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J. Cell Set. 29, 103-116 (1978) 103Printed m Great Britain Company of Biologists Limited igjS

THE DISSOCIATION OF NUCLEAR PROTEINSFROM SUPERHELICAL DNAJEREMY MAX LEVIN, ERICH JOST* AND PETER RICHARDCOOKTh e Sir William Dunn School of Pathology, University of Oxford,South Parks Road, Oxford, OXi 3RE, England, and Europaisches Laboratorium fUr Molekularbiologie,69 Heidelberg 1, Postfach 102209, W. Germany

SUMMARYStructures retaining many of the morphological features of nuclei may be released by gentlylysing human cells in solutions containing non-ionic detergents and high concentrations of salt .These nucleoids contain superhelical DNA. Using a double-labelling procedure we havecompared, at different salt concentrations, th e amounts and types of protein associated withhuman nucleoids containing superhelical or relaxed DNA. We find that the slightly lysine-richhistones (H2A and H2B) but not the arginine-rich histones (H3 and H4) dissociate more slowlyfrom nucleoids containing superhelical DNA than from those containing relaxed DNA. Aprote in of apparent molecular weight of 22000 also binds more t ightly to superhel ica l DNA.We conclude that this protein and the slightly lysine-rich histones transmute free energy ofsupercoiling into binding energy when they bind to superhel ica l DNA.

I N T R O D U C T I O NOne description of the folding of the circular DNA of viruses is based upon adistinction between the right-handed helical turns of the double helix and anysuperhelical turns that might be superimposed upon the duplex turns (Bauer &Vinograd, 1968, 1971). We have been studying DNA conformation in nuclear struc-tures derived by lysing cells in solutions containing non-ionic detergents and highconcentrations of salt. These nucleoids contain all the nuclear RNA and DNA(Colman & Cook, 1977). The nudeoid DNA is supercoiled and the supercoiling maybe released by breaking the DNA with y-rays (Cook & Brazell, 1975, 1976a, b, 1977);(see also Id e, Nakane, Anzai & Andoh , 1975; Benyajati & Worcel, 1976). Nucleoidsisolated in high salt concentrations contain few of the proteins characteristic ofchromatin; when isolated in lower salt concentrations they contain histones (Cook,Brazell & Jost, 1976). Recent evidence suggests that chromatin is composed of sub-units (K ornberg , 1974; Senior, Olins & Olins, 1975; Griffith, 1975) and histones H 3and H4 have been implicated in the primary folding of DNA in the nucleus (Wilkins,Zubay & Wilson, 1959; Richards & Pardon, 1970; Boseley et al . 1976; Camerino-Otero, Solner-Webb & Felsenfeld, 1976). Using a double-labelling procedure, wehave compared the amounts and types of protein bound to human nucleoids con-taining superhelical or relaxed DNA. The comparison indicates that the slightlylysine-rich histones (H2A and H2B), but not the arginine-rich histones (H3 and H4),remain bound more tightly to superhelical DNA than to relaxed DNA.


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10 4 J- M- Levin, E. Jost and P. R. CookMETHODS AND MATERIALSCells and radioactive labelling

He La cells were grown in suspension as described (Cook & Brazell, 1975). Tw o batche s ofcells were labelled simultaneously for 20-24 h> one with [*H]leucine (The RadiochemicalCentre, Amersh am; 100 /tCi/ml; 54000 Ci/mol), the other with [14C]leucine (50/tCi/ml;330 Ci/mol). Cells grown in [14C]leucine multiplied slightly more slowly than in [^HJleucine.Labelled cells were washed 3 times in phosphate-buffered saline (Dulbec co & V ogt, 1954) andresuspended in phosphate-buffered saline at room temp erature.Mixtures used for lysing cells

A num ber of mixtures, adjusted to pH 8 0 at 20 C but containing different salt concen-trations, were used to lyse cells. The final salt concentration of the mixture after the additionof cells is designated in parenthe ses. We neglect the con tribution of the phosphate-bufferedsaline in calculating the final concentration of sodium chloride. Lysis mixtures (19 5 M NaCl),(1-2 M NaCl), (i-o M NaCl) and (0-75 M NaCl) all contained EDTA, Tris and Triton such thatafter adding 1 vol. of phosphate-buffered saline containing cells to 3 vol. of the mixture, the finalconcentrations of the constituents become o-i M, 2 mM and 0-5 % respectively. Lysis mixture(0-4 M NaCl) contained E DT A, Tris, Brij and glycerol such that on addition of 1 vol. ofphosphate-buffered saline containing cells to 3 vol. of the mix ture, the final concen tration ofthe constituents become 01 M, 2 mM, 1 % and 10% respectively.y-irradiation

Sucrose gradients were irradiated immediately after addition of the cells in phosphate-buffered saline to the lysis mixtu re floating on top of the gradien ts (Cook & Brazell, 1975,1976a).Sucrose and glycerol g radients

Nucleoids were isolated free of dissociated proteins after sedimentation through 'isokinetic'sucrose or glycerol gradients (Cook et al. 1976). Sucrose gradients (15-30% sucrose, 4-6 ml,pH 80) contained sodium chloride (0-4, 0-75, 10, 1-2 or 1-95 M), Tris (10 mM) and ED TA(i-o mM). Glycerol gradients (30-50% glycerol, 4'6 ml, pH 8-o) contained NaCl (0-4 M), Tris(10 mM) and E DT A (1 0 mM). 150/tl of a lysis mixture containing the same N aCl concen-tration as the gradient were layered on the gradient, followed by 50 fi\ of phosphate-bufferedsaline containing cells (12-16 x 10' cells/ml). Sometimes ethidium at a final concentration of4 or 16 /tg/ml was added to both lysis mixture and the gradient. 10 or 15 min after the add itionof the cells to the lysis mixture, the gradients were spun at 20 C in an SW 50.1 rotor on aBeckman L2-6sb ultracentrifuge at speeds and times indicated in the legends to the figures.Th e visible aggregates of nucleoids were collected using a 19 G need le inserted throu gh theside of the tube.Two g radients were generally prepared together. Where appropriate the nucleoids from eachgradient were mixed immediately after isolation and the salt concentration lowered to 0-2 Mby dilution in ice-cold 10 mM Tris (pH 80) containing 01 mM phenylmethylsulphonylfluoride.Th e mixed nucleoids were pelleted by centrifugation (Beckman S W 50 .1 rotor ; 40000 rev/min; 15 min; 4 CC) and stored at 70 C.

Analysis of proteinProteins were separated by electrophoresis on polyacrylamide gel slabs containing sodiumdodecyl sulphate (Laemli, 1970). Purified histone fractions (a gift from Dr E. W. Johns) andbovine plasma albumin (Arm our Pharmaceutical Co. Ltd ., Eastbourne) were used as references.Nucleoid proteins were isolated by resuspending nucleoids in 1 % sodium dodecyl sulphate,10 mM Tris (pH 8) and 1 mM phenylmethylsulphonylfluoride. After the DNA had been


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Nuclear proteins and superhelical DNA 105pelleted by centrifugation (Beckman S W 5 0. 1 rotor ; 40000 rev/m in; 3 h; 4 C) the super-natant was dialysed against 3 changes of distilled water containing o-i mM phenylmethyl-sulphonylfluoride and then freeze-dried. The proteins were redissolved in sample buffer con-taining 10 % glycerol, 2 % sodium dodecyl sulphate and o-i M dithiothreitol. Nucleoid proteinwas also prepared for electrophoresis by dissolving nucleoid pellets in 75 fi\ sample buffer andshearing the DNA by passage through a micropipette. Mixtures of protein and sheared DNAgave identical electrophoretic patterns to those given by protein free of DNA. The gels werestained with o-i % Coomassie blue and photographed. Photographic negatives were analysedusing a Joyce-Loebl densitometer.Radioactive profiles of gels were obtained by slicing the gels (slices 1 mm thick) and measuringthe radioactive content of each slice (Cook et al. 1976).The radioactivity due to 'H or 14C in each slice was expressed as a fraction of the totalamount of 3H or 14C in the whole gel. In each slice the ratio of the fractions of each label pro-vides a sensitive index of the relative propo rtions of that label boun d to irradiated and unirr adi-ated nucleoids. Thus flj "1 f bx ' = affinity index- r i

- n _where ax and bm are the amounts of label derived from unirradiated and irradiated nucleoidsrespectively in the xt\\ slice of a gel sliced into n slices. The fraction of label from unirradiatednucleoids is the numerator so that indices above unity characterize proteins present in greaterquantities in unirradiated nucleoids. Where proteins from unirradiated nucleoids isolated inthe presence or absence of ethidium are compared, the fraction of label from the nucleoidsisolated in the absence of ethidium is the numerator. Again indices above unity characterizeproteins present in greater quantities in nucleoids whose DNA is naturally supercoiled.

RESULTSTh e protein content of equal numbers of nucleoids isolated in different salt concen-trations was analysed in polyacrylamide gels containing sodium dodecyl sulphate(Fig. 1). Even in 1-95 M NaCl some proteins (molecular weights > 38000) remainassociated with th e nucleoids (Fig. 1). Since the bind ing of these proteins is unaffectedby radiation or ethidium (see later) they serve as useful reference proteins for com-parison with the other nucleoid proteins. Nucleoids isolated in 0-4 M NaCl containtraces of histone Hi and a number of proteins which are absent in nucleoids isolatedat^higher salt concentrations. As the salt concentration is raised above 0-75 M NaCl,first histones H2A, H2B, and then H 3 and H4 are lost from nucleoids, as is the casewith chromatin (Ohlenbusch, Olivera, Tu an & Davidson, 1967; Henson & Walker,1970); in 1-95 M NaCl the nucleoids contain no histones. A protein which we pro-

visionally call supercoil-binding protein 1 (SB Pi) , of apparent molecular weight22000, is found in nucleoids isolated at or below salt concentrations of i-o M NaCl.We next compared the proteins associated with nucleoids containing DNA ofdifferent conformation. The various nucleoid proteins dissociate at different saltconcentrations so that differences in the relative amounts of these proteins will onlybe detectable under dissociating conditions. We therefore performed 2 kinds ofexperiment at a variety of salt concentrations. In the first, the proteins of nucleoidswith the natural DNA conformation were compared with those of nucleoids irradiatedwith a low dose of y-rays sufficient to remove supercoiling but insufficient to introducemany breaks into DNA.


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106 J. M . Levin, E. Jost and P. R. CookHeLa cells labelled with [3H]leucine were lysed on top of one sucrose gradient andcells labelled with [14C]leucine on top of another. One sucrose gradient was y-irradiated. The nucleoids in both gradients were separated from dissociated proteinby centrifugation and then removed from each of the 2 gradients, mixed and the

BSA H1 SBP1 H2 BH 3 I H 2 A H 4

Migrat ion -Fig. 1. A comparison of the proteins of equal numbers of nucleoids isolated at differentsalt concentrations. Proteins from nucleoids isolated at various salt concentrations inthe absence of ethidium were separated in a polyacrylamide gel (length 10 cm) con-taining sodium dodecyl sulphate, stained with C oomassie blue and the optical densityprofile of the gel determined. The nucleoids were prepared from cells grown in[JVfe-*H]thymidine (56000 Ci/mol; o#o2 /iCi/ml) for 24 h and equivalent amounts ofradioactivity were applied to each gel. Reference proteins, bovine plasma albumin(BSA) and histones (Hi, H2A, H2B, H3 and H4) were run in adjacent channels.Proteins from nucleoids isolated in: A, 0 4 M NaCl; B, 0-75 MNaCl; C, f o M NaCl;D, 1-2 M NaCl; and E, 1-95 M NaCl were analysed in this way.

mixtu re subjected to electrophoresis in polyacrylamide gels containing sodium dodecylsulphate. The relative proportions of 3H and 14C in the gel reflect the relative pro-portions of protein derived from irradiated or unirradiated nucleoids. We derive theaffinity index - a measure of the binding to superhelical DNA relative to the relaxedform - by normalizing the radioactivities (see Materials and methods); indices aboveunity characterize proteins present in greater amounts in unirradiated nucleoids thanirradiated nucleoids.In the second kind of experiment the proteins of nucleoids with the natural DNA


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Nuclear proteins an d superhelical DNA 107conformation were compared with those of unirradiated nucleoids isolated in thepresence of ethidium. Ethidium intercalation unwinds the double helix removing(negative) superhelical turns (e.g. in I-OM NaCl, 4/tg/ml ethidium removes super-coiling from nucleoid DNA). At higher concentrations (i.e. i6/fg/ml in i-o M NaCl)supercoils of the opposite sense to those intitially present are induced (Cook & Brazell,1975). Cells labelled with pHJleucine were layered on one sucrose gradient and 14 C-labelled cells on another; neither gradient was irradiated but one contained ethidium.After the 2 kinds of nucleoids had been isolated and mixed, their proteins were sub-jected to electrophoresis in gels. Again affinity indices above unity characterizeproteins remaining more tightly bound to nucleoids with the natural superhelicalconformation.

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jeremy max levin, erich jost and peter richard cook- the dissociation of nuclear proteins from...

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